Anti-diabetic and renal protective properties of Stevia rebaudiana

Stevia

Dr Naveen S

Department of Applied Nutrition,

Defence Food Research Laboratory,

Mysore-570011. Karnataka, India.

Telephone: +91-821-2579488

Fax: +91-821-2473468

E-mail: naveen.dfrl@gmail.com

Is an herb native to Central and South America. Also known

as sugar leaf, honey leaf or sweet leaf, it's been traditionally used in

Paraguay to sweeten yerba mate tea and as a medicinal herb to

treat a variety of health conditions. Composed of aglycone and

glycoside.

Stevia Leaves

Liquid Extracts

Powdered Extracts

Stevia extractsO glucose

O

glucose

glucose

O

glucose

H

H

CH3CH2

O

CH3

O

• Hot-water extracts from the leaves of the Brazilian shrub Stevia rebaudiana

• Contains many steviol glycosides, with highest percentage of stevioside and rebaudioside A

• JECFA ADI = 4 mg/kg/day, as steviol equivalents• Rebaudoside A preparation permitted in U.S. for food use

glucoseO

glucose

O

glucose

H

H

CH3CH2

O

CH3

O

Rebaudioside A

Stevioside

Stevia extracts: Metabolism• Glucosides not absorbed • Steviol glycosides hydrolysed to

steviol by gut microflora – Rate depends on number of

glucose moieties attached to steviol backbone

• Steviol absorbed in large intestine, glucuronidated and excreted– Rats: faeces– Humans: urine

R1O

H

H

CH3CH2

O

CH3

OR2

Steviol Backbone

FDA approved Stevia

The Food and Drug Administration has declared the herb stevia

safe for use in foods and beverages, which allows Coca-Cola Co.,

PepsiCo Inc. and other companies to market it in a variety of

products.

Coke introduced a reduced-calorie version of Sprite, called Sprite

Green, and some Odwalla juice drinks with stevia extract.

Pepsi has SoBe Lifewater, and an orange-juice drink called

Trop50, containing half the calories and sugar of orange juice.

Stevioside approval Country list

• Japan United States• Korea Canada • China Australia • India New-Zealand • Indonesia Turkey• Taiwan Thailand• South America Russia• Swizerland

Specification ComparisonSucrose Artificial

SweetenersSteviosides Benefit

Type Natural Artificial Natural Safe

Sweetness Level

1time 300 times 300 times Reduce RM cost

Heat stability 90 Celsius 200Celsius Suitable for cooking

Acid-base resistance

High Low High Safe No taste changing

Safety High Carcinogen Anti-carcinogenic

Preservatives No No Yes Increase shelf-lifeReduce cost of packaging

Health benefits

DiabetesObesity

Nerve problem blood sugar blood pressureWeight control

Value –added product

Calories high low no Low sugar food and drink

Product ApplicationProducts

itemsSubstitute dosage for

sugarAdvantages

Beverage 10-50%

(1) Improve taste (2) Reduce high sweetness from

sugar(3) It can be used for people who can

not have much sugar.

MedicineAccording to the

requirements

(1) Reduce the cost (2) Improve the health care function (3) Improve the taste

Cold food 10-25% (1) Improve the sweetness.

Caned food 20-50%

(1) The stevia liquid is very clear and its taste is fresh.

(2) Reduce the much sweetness from sugar

(3) Prolong the shelf-time

Aquatic food 30-50%(1) It can stop the browning reaction

and moldy.

Product Application

Products items

Substitute dosage for sugar

Advantages

Beer 10-40%(1) Make the taste better.(2) It can bring much more foam;

make the foam lasting and white.

Fruit wine 50%(1) It can improve the wine’s scent (2) Reduce the stickiness, improve

the fresh taste.

liqueur 10-20%(1) It can remove the hot taste from the wine

STZ

STZ

RATSSACRIFICED

Dt1

Group 5Test 2

Group 4STZ + Test 1

STZ

Group 1Control

Group 2STZ

Group 3 Test Contrl

Dt2

Experimental ProtocolParameters studied: (Liver / Heart / Kidney) Lipid peroxidation:

MDA Hydroperoxides Conjugated dienes

Antioxidant metabolites or molecules: GSH Vitamin C

Antioxidant enzymes: SOD GST Catalase GSHPx GSSGR G-6-PDH

Serum / Plasma Glucose Lipid profile SGOT SGPT

Liver 1. Glycogen

Rats were segregated randomly into 8 groups of 10 rats each. Groups 1 and 2 were fed with the control diet; group 3 and 4 with 4.0% stevia leaves

powder incorporated diet (4.0g leaf powder in 96g dry diet); Group 5 and 6 with equivalent amount of polyphenols extract (through force

feeding); Group 7 and 8 with equivalent amount of fiber extracted from 4 g of stevia leaves

powder respectively for 5 weeks, All the rats were fed ad libitum with free access to water. Food intake and weight gain were monitored weekly. One week prior to sacrifice, all even groups were given a single dose of STZ

(60mg/kg body weight, i.p. using a 5% solution of freshly prepared streptozotocin in

0.1 M citrate buffer pH 4.5). Rats were sacrificed under mild anesthesia using anesthetic ether, after which

organs / tissues were quickly excised and stored in liquid nitrogen until analyses

(completed immediately within a week).

Antioxidant property of stevia leaves was estimated and compared with other commercial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and Tertiary butyl hydroquinone (TBHQ).

Stevia leaves were having better antioxidant properties such as free radical scavenging and inhibition of lipid per-oxidation

Thiobarbituric acid reaction substances, conjugated dienes and hydroperoxides; products of lipid peroxidation is a measure of oxidative stress in liver.

The levels of malondialdehyde, conjugated dienes and hydroperoxides increased upon injection of STZ, by three times compared to untreated group.

Antioxidant activity (in-vitro) of Stevia leaves (n=3)

Antioxidant properties of SteviaEC50 (mg)

Stevia BHA* BHT* TBHQ*

1. Lipid per-oxidation inhibition property 2.6 + 0.05 2.9 + 0.04 2.2 + 0.02 3.8 + 0.02

2. Free radical scavenging property

(DPPH scavenging)10.6 + 1.91 13.0 + 1.72 43.2 + 2.84 8.6 + 0.11

*Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and Tertiary butyl

hydroquinone (TBHQ).

Peroxidation was reduced significantly in rats prefed with stevia leaves powder and polyphenols extracted to 30-25% in liver compared to diabetic group.

Pre-feeding stevia however did not alter the levels of antioxidants in plasma. STZ administration resulted in significant elevation of hepatic antioxidant enzymes SOD to 33% and

catalase to 74% compared to control group, when rats were prefed with stevia leaves powder and polyphenols extracted the enzyme activity significantly reduced by 15-25% and 40-55% respectively compared to diabetic group.

Hepatic phase I GSHPx activity was reduced by 52% on STZ treatment, which on pre-feeding with stevia leaves powder and polyphenols extracted the activity increased to 90-84% compared to diabetic group. No significant change was seen in extracted fiber fed group.

Effect of Stevia on lipid peroxidation in liver in treated rats (n=8)TBARS

(x 10-10 moles / g)Hydroperoxides(x 10-4 moles / g)

Conjugated dienes

(x 10-4 moles / g)

Control 0.06 + 0.01a 0.07 + 0.02a 0.12 + 0.02a

Streptozotocin 0.16 + 0.03* 0.17 + 0.03* 0.24 + 0.06*Stevia leaves Powder 0.08 + 0.02a 0.11 + 0.03 0.09 + 0.02a

+ Stevia leaves Powder 0.12 + 0.03* 0.10 + 0.02a 0.17 + 0.04a

Stevia leaves polyphenols 0.07 + 0.01a 0.09 + 0.02 0.10 + 0.02a

+ Stevia leaves polyphenols 0.11 + 0.02* 0.08 + 0.01a 0.14 + 0.03a

Stevia leaves fiber 0.05 + 0.01a 0.08 + 0.02a 0.15 + 0.03a

+ Stevia leaves fiber 0.17 + 0.02* 0.15 + 0.03* 0.25 + 0.05*

Note: Significance is defined as p < 0.05. Superscript ‘*’ indicates the value is significantly different in comparison with Control (P<0.05) and superscript ‘a’ indicates the value is significantly different in comparison with the treated group (STZ) at P<0.05.

Treatment of animals with STZ, a known diabetogen (Marianna et al., 2006) led to polydypsia, polyphagia and polyurea.

Food consumption increased from 120g to 300g/week, while water intake almost doubled from 20ml/day to 40ml/day. Urine output

increased from 8.0ml to 15ml/day.

The body weight which was 306g on the day of injection of STZ was 240g at the weekend i.e, a loss of 30% in one week.

Pre-feeding the rats with stevia leaves powder and polyphenols extracted before injecting STZ prevented these changes significantly; food

consumption was brought down by 20-16%, water consumption by 55-50%, urine output by 55-47% and loss in body weight was limited to

5-10g/week by stevia leaves powder and polyphenols extracted.

The diabetic rats showed a significant increase in the blood glucose level when compared to the normal rats (P<0.05).

Stevia leaves powder and its polyphenol extract reduced the elevated levels of blood glucose by 50-30% compared to the untreated

diabetic control (P<0.05). but there was no reduction in blood glucose level in stevia fiber fed rats.

In the STZ group, the serum insulin level was lower than that of the normal group (P<0.05).

Effect of stevia on Anti-diabetic action in treated rats (n=8)  Control Stevia STZ STZ + Stevia

leaves powder

Stevia

polyphenol

STZ + Stevia

polyphenol

Stevia fiber STZ + Stevia

fiber

Weight gain (g/week) 25.8 + 9.70 25.7 + 14.3 6.8 + 1.8*a 11.7 + 1.60*a 21.5 + 11.4 10.3 + 0.95*a 22.5 + 9.12 6.0 + 2.1*a

Food intake (g/day) 9.4 + 3.7 10.2 + 3.00 13 + 1.9 11 + 3.3 9.8 + 2.50 10.1 + 2.3 9.1 + 2.6 11 + 1.1

Water intake (ml/day) 27.9 + 16.1 20.8 + 13.3 59.5 + 28.4*a 42.6 + 23.3*a 22.1 + 16.1 52.6 + 25.2*a 25.1 + 14.3 55.2 + 22.1*a

Urine volume (ml/24hrs) 39.0 + 17.7 31.1 + 14.7 98.4 + 27.9*a 57.2 + 16.8*a 28.3 + 11.6 53.1 + 11.5*a 42.2 + 15.1 89.9 + 17.8*a

Blood glucose (mg/dl) 109.2 + 29.60 99.8 + 22.0 356.3 + 65.40*a 228.2 + 22.10*a 101.4 + 17.2 129.2 + 12.50*a 99.3 + 10.46 342.0 + 42.50*a

Effect of stevia on kidney functions in treated rats (n=8) 

Control Stevia STZ + Stevia leaves

powder

Stevia

polyphenol

+ Stevia

polyphenol

Stevia fiber + Stevia fiber

Plasma Creatine0.47 + 0.11 0.40 + 0.14 0.57 + 0.02 0.53 + 0.12 0.50 + 0.22 0.45 + 0.09 0.48 + 0.08 0.60 + 0.10

Urinary Creatine0.071 + 0.003 0.082 + 0.014 0.058 + 0.002*a 0.062 + 0.002*a 0.080 + 0.01 0.059 + 0.008*a 0.069 + 0.01 0.059 + 0.004*a

GFR(ml/min/kg body wt)

5.55 + 0.24a 6.42 + 0.32* 4.56 + 0.77a 4.89 + 0.18a 6.01 + 0.12* 4.95 + 0.06a 5.49 + 0.11a 4.44 + 0.20a

A slight reduction, though not significantly; in the kidney weight was observed in stevia leaves

powder and polyphenols extracted groups, which increased on STZ treatment.

Also there was an increase in GFR in STZ treated rats and was reduced on pre-feeding with stevia

leaves powder and polyphenols extracted.

The data on the effect of stevia on kidney antioxidant enzymes viz. G-6-PDH, and detoxifying enzyme

-glutamyl transpeptidase showed administration of STZ resulted in decreased G-6-PDH and

increased g-glutamyl transpeptidase activity.

The STZ-induced elevation in renal G-6-PDH was brought down by stevia leaves powder but both the

enzyme activities were bought down by the polyphenols extracted.

No significant change was seen in extracted fiber fed group.

Effect of Stevia leaves on hepatic marker enzymes of STZ treated rats (n=8)

Normal values of ALT and AST in serum for rats is 46-80 and 18-30IU/dl respectively (Kalyanasundarm, 1985).

Serum ALT and AST levels were significantly increased in STZ treated animals by 89 and 42% compared to control group (Fig. 4).

Pre-feeding of stevia leaves powder and extracted polyphenols to diabetic animals reduced the activity to 45-38 and 13-6% compared to diabetic group respectively.

No significant change was seen in extracted fiber fed group.

0

10

20

30

40

50

60

70

80

Control Stz Stz + Stevia Stevia Stz +polyphenol

polyphenol Stz + f iber f iber

IU/d

lSGOT

SGPT

The identification of major polyphenols was based on comparison of their retention times with those of reference compounds and their elution order on reversed-phase C18 columns.

Peak assignment was confirmed by mass spectrometry. Major compounds identified are dicaffeoylquinic acid, chlorogenic acid, Quercetin 3-O-xyloside,

Apigenin-7-O-glucoside, 3,4-Dimethoxycinnamic acid, Luteolin 7-O-rutinoside, Caffeic acid etc Stevia was found to oppose the hyperglycemic actions in the diabetic treated rats. Previous studies had also noted the importance of hypoglycemic components of Stevia is because

of Rebaudioside A and stevioside that are concentrated in leaves. In our study none of the compounds identified in the polyphenol extract was Rebaudioside A or

stevioside, since the mass was not matching with compounds detected. This explains the involvement of polyphenolic compounds in preventing diabetic and its

complications caused by streptozotocin

Phenolic compounds identification in Stevia leaves

2x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

DAD1 - A: Signal A DFRL Sample-S_001-r001.d

0.9

52

1.0

14

1.0

66

1.1

24

1.1

91

1.2

27

1.4

52 1.5

48

2.9

64

3.0

92

3.2

16

4.7

50

6.1

34

6.8

21

7.1

65

7.3

69

7.5

52

7.6

55

8.3

25

8.5

77

8.6

21

8.7

93

8.9

04

8.9

75

9.1

10

9.1

66

9.2

30

9.4

05

9.6

21

9.9

02

10.1

90

10.2

89

11.0

48

11.2

33

11.3

50

11.4

47

12.2

58

14.0

26

16.8

86

Response Units vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18

5x10

0

0.25

0.5

0.75

1

1.25

1.5

-ESI Product Ion:4 (2.866 min) Frag=80.0V CID@8.0 (353.0 -> **) DFRL Sample-S_MSMS_001.d

191.0

179.0 352.9 135.1

5x10

0

0.25

0.5

0.75

1

1.25

1.5

-ESI Product Ion:4 (3.210 min) Frag=80.0V CID@8.0 (353.0 -> **) DFRL Sample-S_MSMS_001.d

191.1

353.0 160.9 178.8

4x10

0

1

2

3

4

-ESI Product Ion:4 (4.658 min) Frag=80.0V CID@8.0 (353.0 -> **) DFRL Sample-S_MSMS_001.d

173.0

191.0 135.0

353.1

155.0 111.0

Counts vs. Mass-to-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360

1-O-Caffeoylquinic acid or 1-Chlorogenic acid

3-O-Caffeoylquinic acid or 3-Chlorogenic acid

4-O-Caffeoylquinic acid or 4-Chlorogenic acid

CONCLUSIONS All these results are quite interesting and very encouraging.

One single plant could have multifaceted benefits.

However, the study brings forth many questions which need further thorough

investigation, like the mechanism of action of stevia leaves powder inactivating

the STZ in the blood itself, or does it prevent its transport across into pancreatic

and hepatic cells? Whether the extract simply mops off the reactive oxygen

species or is it a chain breaking antioxidant? Whether the prevention of the liver

damage seen with stevia leaves is the result of its antioxidant property or does it

induce the activity of any other liver enzymes? The plant seems to be worth

investigating further and could be an excellent source for nutraceuticals.

In conclusion, polyphenols present in stevia leaves are mainly responsible for its

multi-beneficial properties.

Thank you for attention